Synaptically driven endocannabinoid release requires Ca2+- assisted metabotropic glutamate receptor subtype 1 to phospholipase C β4 signaling cascade in the cerebellum

金沢大学医薬保健研究域保健学系Endocannabinoids mediate retrograde signaling and modulate synaptic transmission in various regions of the CNS. Depolarization-induced elevation of intracellular Ca2+ concentration causes endocannabinoid-mediated suppression of excitatory/inhibitory synaptic transmission. Activation of G q/11-coupled receptors including group I metabotropic glutamate receptors (mGluRs) also causes endocannabinoid-mediated suppression of synaptic transmission. However, precise mechanisms of endocannabinoid production initiated by physiologically relevant synaptic activity remain to be determined. To address this problem, we made whole-cell recordings from Purkinje cells (PCs) in mouse cerebellar slices and examined their excitatory synapses arising from climbing fibers (CFs) and parallel fibers (PFs). We first characterized three distinct modes to induce endocannabinoid release by analyzing CF to PC synapses. The first mode is strong activation of mGluR subtype 1 (mGluR1)-phospholipase C (PLC) β4 cascade without detectable Ca 2+ elevation. The second mode is Ca2+ elevation to a micromolar range without activation of the mGluR1-PLC/34 cascade. The third mode is the Ca2+-assisted mGluR1-PLCβ4 cascade that requires weak mGluR1 activation and Ca2+ elevation to a submicromolar range. By analyzing PF to PC synapses, we show that the third mode is essential for effective endocannabinoid release from PCs by excitatory synaptic activity. Furthermore, our biochemical analysis demonstrates that combined weak mGluR1 activation and mild depolarization in PCs effectively produces 2-arachidonoylglycerol (2-AG), a candidate of endocannabinoid, whereas either stimulus alone did not produce detectable 2-AG. Our results strongly suggest that under physiological conditions, excitatory synaptic inputs to PCs activate the Ca2+-assisted mGluR1-PLCβ4 cascade, and thereby produce 2-AG, which retrogradely modulates synaptic transmission to PCs. Copyright © 2005 Society for Neuroscience.This work was supported by Grants-in-Aid for Scientific Research and Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Sports, Culture, Science and Technology ofJapan. This work was alsosupported by theJapan Society for the Promotion of Science (JSPS) and the Toyota RIKEN Foundation. T.M. was a recipient of JSPS Research Fellowships for Young Scientists and the Research Aid of Inoue Foundation for Science. We thank S. Arai for 2-AG estimation and Drs. K. Hashimoto and T. Tabata for comments on this work